RESUMO
An early step of target validation in antimicrobial drug discovery is to prove that a gene coding for a putative target is essential for pathogen's viability. However, little attention has been paid to demonstrate the causal links between gene essentiality and a particular protein function that will be the focus of a drug discovery effort. This should be considered an important step in target validation since a growing number of proteins are found to exhibit multiple and unrelated tasks. Here, we show that the Mycobacterium tuberculosis (Mtb) folB gene is essential and that this essentiality depends on the dihydroneopterin aldolase/epimerase activities of its protein product, the FolB protein from the folate biosynthesis pathway. The wild-type (WT) MtFolB and point mutants K99A and Y54F were cloned, expressed, purified and monitored for the aldolase, epimerase and oxygenase activities using HPLC. In contrast to the WT MtFolB, both mutants have neither aldolase nor epimerase activities in the conditions assayed. We then performed gene knockout experiments and showed that folB gene is essential for Mtb survival under the conditions tested. Moreover, only the WT folB sequence could be used as a rescue copy in gene complementation studies. When the sequences of mutants K99A or Y54F were used for complementation, no viable colonies were obtained, indicating that aldolase and/or epimerase activities are crucial for Mtb survival. These results provide a solid basis for further work aiming to develop new anti-TB agents acting as inhibitors of the aldolase/epimerase activities of MtFolB.
Assuntos
Aldeído Liases/antagonistas & inibidores , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Descoberta de Drogas/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Aldeído Liases/genética , Aldeído Liases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Cromatografia Líquida de Alta Pressão , Genes Essenciais/genética , Teste de Complementação Genética/métodos , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/genética , Terapia de Alvo Molecular/métodos , Mutação de Sentido Incorreto , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Reprodutibilidade dos Testes , Especificidade por Substrato , Espectrometria de Massas em Tandem , Tuberculose/tratamento farmacológico , Tuberculose/microbiologiaRESUMO
The enzymes of the shikimate pathway represent potential molecular targets for the development of non-toxic antimicrobial agents and anti-parasite drugs. One of the most promising of these enzymes is shikimate kinase (EC 2.7.1.71), which is responsible for the fifth step in the shikimate pathway. This enzyme phosphorylates shikimic acid to yield shikimate-3-phosphate, using ATP as a substrate. In this work, the conformational dynamics of the shikimate kinase from Mycobacterium tuberculosis was investigated in its apostate in solution. For this study, the enzyme was subjected to a gradient of temperatures from 15°C to 45°C in the presence or absence of deuterium oxide, and the amide H/D exchange was monitored using ESI-mass spectrometry. We observed: i) the phosphate binding domain in the apo-enzyme is fairly rigid and largely protected from solvent access, even at relatively high temperatures; ii) the shikimate binding domain is highly flexible, as indicated by the tendency of the apo-enzyme to exhibit large conformational changes to permit LID closure after the shikimate binding; iii) the nucleotide binding domain is initially conformationally rigid, which seems to favour the initial orientation of ADP/ATP, but becomes highly flexible at temperatures above 30°C, which may permit domain rotation; iv) part of the LID domain, including the phosphate binding site, is partially rigid, while another part is highly flexible and accessible to the solvent.
Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Sequência de Aminoácidos , Antituberculosos/química , Antituberculosos/uso terapêutico , Medição da Troca de Deutério , Óxido de Deutério/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização por Electrospray , Temperatura , Tuberculose/tratamento farmacológicoRESUMO
The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, infects one-third of the world population. TB remains the leading cause of mortality due to a single bacterial pathogen. The worldwide increase in incidence of M. tuberculosis has been attributed to the high proliferation rates of multi and extensively drug-resistant strains, and to co-infection with the human immunodeficiency virus. There is thus a continuous requirement for studies on mycobacterial metabolism to identify promising targets for the development of new agents to combat TB. Singular characteristics of this pathogen, such as functional and structural features of enzymes involved in fundamental metabolic pathways, can be evaluated to identify possible targets for drug development. Enzymes involved in the pyrimidine salvage pathway might be attractive targets for rational drug design against TB, since this pathway is vital for all bacterial cells, and is composed of enzymes considerably different from those present in humans. Moreover, the enzymes of the pyrimidine salvage pathway might have an important role in the mycobacterial latent state, since M. tuberculosis has to recycle bases and/or nucleosides to survive in the hostile environment imposed by the host. The present review describes the enzymes of M. tuberculosis pyrimidine salvage pathway as attractive targets for the development of new antimycobacterial agents. Enzyme functional and structural data have been included to provide a broader knowledge on which to base the search for compounds with selective biological activity.
Assuntos
Mycobacterium tuberculosis/enzimologia , Pirimidinas/metabolismo , Citidina Desaminase/metabolismo , Mycobacterium tuberculosis/metabolismo , Núcleosídeo-Difosfato Quinase/metabolismo , Núcleosídeo-Fosfato Quinase/metabolismo , Nucleotídeo Desaminases/metabolismo , Pentosiltransferases/metabolismo , Pirimidina Fosforilases/metabolismo , Pirofosfatases/metabolismo , Timidilato Sintase/metabolismoRESUMO
Millions of deaths worldwide are caused by the aetiological agent of tuberculosis, Mycobacterium tuberculosis. The increasing prevalence of this disease, the emergence of drug-resistant strains, and the devastating effect of human immunodeficiency virus coinfection have led to an urgent need for the development of new and more efficient antimycobacterial drugs. The modern approach to the development of new chemical compounds against complex diseases, especially the neglected endemic ones, such as tuberculosis, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a specific target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, and (iii) the development of compounds with selective toxicity. The present review describes the enzymes of the purine salvage pathway in M. tuberculosis as attractive targets for the development of new antimycobacterial agents. Enzyme kinetics and structural data have been included to provide a thorough knowledge on which to base the search for compounds with biological activity. We have focused on the mycobacterial homologues of this pathway as potential targets for the development of new antitubercular agents.
Assuntos
Mycobacterium tuberculosis/enzimologia , Purinas/metabolismo , 5'-Nucleotidase/metabolismo , Adenosina Desaminase/metabolismo , Adenosina Quinase/metabolismo , Adenilossuccinato Liase/metabolismo , Adenilossuccinato Sintase/metabolismo , IMP Desidrogenase/metabolismo , Mycobacterium tuberculosis/metabolismo , N-Glicosil Hidrolases/metabolismo , Núcleosídeo-Fosfato Quinase/metabolismo , Pentosiltransferases/metabolismo , Purina-Núcleosídeo Fosforilase/metabolismoRESUMO
BACKGROUND AND OBJECTIVE: Purine nucleoside phosphorylase (PNP) is an enzyme that catalyzes the reversible phosphorolysis of purine nucleosides, playing a key role in the purine salvage pathway. Activated T cells seem to rely heavily on PNP to remain functionally active and are particularly sensitive to PNP deficiency. The role of PNP in periodontal tissues has not been characterized thus far. The aim of this study therefore was to assess the activity and expression of PNP in the gingival tissues of periodontitis patients. MATERIAL AND METHODS: Ten patients consecutively admitted for treatment had their periodontal clinical variables recorded and their gingival crevicular fluid collected. After periodontal treatment the patients were seen once a month for plaque and bleeding control, and had their periodontal variables recorded and gingival crevicular fluid collected at 90 and 180 d. Purine nucleoside phosphorylase-specific activity was assessed using a spectrophotometer through the addition of the PNP substrate analog 2-amino-6mercapto-7-methyl purine riboside to the gingival crevicular fluid. In parallel, PNP expression was assessed by immunohistochemistry and real-time PCR in gingival biopsies and cell culture. RESULTS: Purine nucleoside phosphorylase activity was higher in the gingival crevicular fluid of periodontally diseased sites, which was positively correlated with improvements of the clinical variables. Treatment of periodontal disease induced a striking decrease of PNP activity in periodontally diseased sites. Expression of PNP was more pronounced in mononuclear cells and endothelial cells of the gingiva, and the mRNA levels were 5.7-fold higher in inflamed tissues compared with control samples. CONCLUSION: Purine nucleoside phosphorylase activity and expression are upregulated in periodontally diseased sites and can be detected in the gingival crevicular fluid.
Assuntos
Periodontite Agressiva/enzimologia , Periodontite Crônica/enzimologia , Líquido do Sulco Gengival/enzimologia , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , Adulto , Idoso , Periodontite Agressiva/terapia , Linfócitos T CD4-Positivos/enzimologia , Periodontite Crônica/terapia , Regulação Enzimológica da Expressão Gênica , Gengiva/enzimologia , Humanos , Memória Imunológica , Pessoa de Meia-Idade , Distribuição Normal , Estatísticas não Paramétricas , Regulação para CimaRESUMO
This work describes for the first time a model of Purine Nucleoside Phosphorylase from Listeria monocytogenes (LmPNP). We modeled the complexes of LmPNP with ligands in order to determine the structural basis for specificity. Comparative analysis of the model of LmPNP allowed identification of structural features responsible for ligand affinities.
Assuntos
Biologia Computacional , Listeria monocytogenes/enzimologia , Purina-Núcleosídeo Fosforilase/química , Sequência de Aminoácidos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/metabolismo , Sítios de Ligação , Desenho de Fármacos , Humanos , Ligantes , Listeria monocytogenes/efeitos dos fármacos , Listeriose/tratamento farmacológico , Modelos Moleculares , Estrutura Terciária de Proteína , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Purina-Núcleosídeo Fosforilase/metabolismo , Especificidade por SubstratoRESUMO
The rate at which knowledge about genomic sequences and their protein products is produced is increasing much faster than the rate of 3-dimensional protein structure determination by experimental methods, such as X-ray diffraction and nuclear magnetic resonance. One of the major challenges in structural bioinformatics is the conversion of genomic sequences into useful information, such as characterization of protein structure and function. Using molecular dynamics (MD) simulations, we predicted the 3-dimensional structure of an artificially designed three- alpha -helix bundle, called A3, from a fully extended initial conformation, based on its amino acid sequence. The MD protocol enabled us to obtain the secondary, in 1.0 ns, as well as the supersecondary and tertiary structures, in 4.0-10.0 ns, of A3, much faster than previously described for a similar protein system. The structure obtained at the end of the 10.0-ns MD simulation was topologically a three-alpha-helix bundle.
Assuntos
Simulação por Computador , Modelos Moleculares , Proteínas/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , SolventesRESUMO
Tuberculosis (TB) and Malaria are neglected diseases, which continue to be major causes of morbidity and mortality worldwide, killing together around 5 million people each year. Mycolic acids, the hallmark of mycobacteria, are high-molecular-weight alpha-alkyl, beta-hydroxy fatty acids. Biochemical and genetic experimental data have shown that the product of the M. tuberculosis inhA structural gene (InhA) is the primary target of isoniazid mode of action, the most prescribed anti-tubercular agent. InhA was identified as an NADH-dependent enoyl-ACP(CoA) reductase specific for long-chain enoyl thioesters and is a member of the Type II fatty acid biosynthesis system, which elongates acyl fatty acid precursors of mycolic acids. M. tuberculosis and P. falciparum enoyl reductases are targets for the development of anti-tubercular and antimalarial agents. Here we present a brief description of the mechanism of action of, and resistance to, isoniazid. In addition, data on inhibition of mycobacterial and plasmodial enoyl reductases by triclosan are presented. We also describe recent efforts to develop inhibitors of M. tuberculosis and P. falciparum enoyl reductase enzyme activity.
Assuntos
Antimaláricos/administração & dosagem , Antituberculosos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Desenho de Fármacos , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Animais , Antimaláricos/síntese química , Antituberculosos/síntese química , Sistemas de Liberação de Medicamentos/tendências , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/antagonistas & inibidores , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/química , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/síntese química , HumanosRESUMO
Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and thus drugs that inhibit human PNP activity have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Besides, the purine salvage pathway is the only possible way for apicomplexan parasites to obtain the building blocks for RNA and DNA synthesis, which makes PNP from these parasites an attractive target for drug development against diseases such as malaria. Hence, a number of research groups have made efforts to elucidate the mechanism of action of PNP based on structural and kinetic studies. It is conceivable that the mechanism may be different for PNPs from diverse sources, and influenced by the oligomeric state of the enzyme in solution. Furthermore, distinct transition state structures can make possible the rational design of specific inhibitors for human and apicomplexan enzymes. Here, we review the current status of these research efforts to elucidate the mechanism of PNP-catalyzed chemical reaction, focusing on the mammalian and Plamodium falciparum enzymes, targets for drug development against, respectively, T-Cell- and Apicomplexan parasites-mediated diseases.
Assuntos
Apicomplexa/enzimologia , Sistemas de Liberação de Medicamentos/métodos , Infecções por Protozoários/enzimologia , Purina-Núcleosídeo Fosforilase/metabolismo , Linfócitos T/enzimologia , Animais , Apicomplexa/patogenicidade , Humanos , Infecções por Protozoários/tratamento farmacológico , Infecções por Protozoários/parasitologia , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Linfócitos T/parasitologiaRESUMO
The aetiological agent of tuberculosis (TB), Mycobacterium tuberculosis, is responsible for millions of deaths annually. The increasing prevalence of the disease, the emergence of multidrug-resistant strains, and the devastating effect of human immunodeficiency virus co-infection have led to an urgent need for the development of new and more efficient antimycobacterial drugs. Since the shikimate pathway is present and essential in algae, higher plants, bacteria, and fungi, but absent from mammals, the gene products of the common pathway might represent attractive targets for the development of new antimycobacterial agents. In this review we describe studies on shikimate pathway enzymes, including enzyme kinetics and structural data. We have focused on mycobacterial shikimate pathway enzymes as potential targets for the development of new anti-TB agents.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Desenho de Fármacos , Mycobacterium tuberculosis/enzimologia , Ácido Chiquímico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/síntese química , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Ácido Chiquímico/antagonistas & inibidores , Ácido Chiquímico/síntese química , Transdução de Sinais/fisiologiaRESUMO
The rate at which knowledge about genomic sequences and their protein products is produced is increasing much faster than the rate of 3-dimensional protein structure determination by experimental methods, such as X-ray diffraction and nuclear magnetic resonance. One of the major challenges in structural bioinformatics is the conversion of genomic sequences into useful information, such as characterization of protein structure and function. Using molecular dynamics (MD) simulations, we predicted the 3-dimensional structure of an artificially designed three-alpha-helix bundle, called A3, from a fully extended initial conformation, based on its amino acid sequence. The MD protocol enabled us to obtain the secondary, in 1.0 ns, as well as the supersecondary and tertiary structures, in 4.0-10.0 ns, of A3, much faster than previously described for a similar protein system. The structure obtained at the end of the 10.0-ns MD simulation was topologically a three-alpha-helix bundle.
Assuntos
Simulação por Computador , Modelos Moleculares , Proteínas/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , SolventesRESUMO
Tuberculosis resurged in the late 1980s and now kills more than 2 million people a year. The reemergence of tuberculosis as a potential public health threat, the high susceptibility of human immunodeficiency virus-infected persons to the disease, and the proliferation of multi-drug-resistant (MDR) strains have created much scientific interest in developing new antimycobacterial agents to both treat Mycobacterium tuberculosis strains resistant to existing drugs, and shorten the duration of short-course treatment to improve patient compliance. Bacterial cell-wall biosynthesis is a proven target for new antibacterial drugs. Mycolic acids, which are key components of the mycobacterial cell wall, are alpha-alkyl, beta-hydroxy fatty acids, with a species-dependent saturated "short" arm of 20-26 carbon atoms and a "long" meromycolic acid arm of 50-60 carbon atoms. The latter arm is functionalized at regular intervals by cyclopropyl, alpha-methyl ketone, or alpha-methyl methylethers groups. The mycolic acid biosynthetic pathway has been proposed to involve five distinct stages: (i) synthesis of C20 to C26 straight-chain saturated fatty acids to provide the alpha-alkyl branch; (ii) synthesis of the meromycolic acid chain to provide the main carbon backbone, (iii) modification of this backbone to introduce other functional groups; (iv) the final Claisen-type condensation step followed by reduction; and (v) various mycolyltransferase processes to cellular lipids. The drugs shown to inhibit mycolic acid biosynthesis are isoniazid, ethionamide, isoxyl, thiolactomycin, and triclosan. In addition, pyrazinamide was shown to inhibit fatty acid synthase type I which, in turn, provides precursors for fatty acid elongation to long-chain mycolic acids by fatty acid synthase II. Here we review the biosynthesis of mycolic acids and the mechanism of action of antimicrobial agents that act upon this pathway. In addition, we describe molecular modeling studies on InhA, the bona-fide target for isoniazid, which should improve our understanding of the amino acid residues involved in the enzyme's mechanism of action and, accordingly, provide a rational approach to the design of new drugs.
Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Ácidos Micólicos/antagonistas & inibidores , Antituberculosos/química , Antituberculosos/uso terapêutico , Parede Celular/metabolismo , Humanos , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/química , Relação Estrutura-Atividade , Tuberculose/tratamento farmacológico , Tuberculose/prevenção & controleRESUMO
Tuberculosis (TB) resurged in the late 1980s and an estimated 1.87 million people died of TB in 1997. The reemergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons, and the proliferation of multidrug-resistant strains have created a need to develop new antimycobacterial agents. The existence of a shikimate pathway has been predicted by the determination of the genome sequence of Mycobacterium tuberculosis. The M. tuberculosis aroK-encoded shikimate kinase and aroA-encoded 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase were cloned and the enzymes overexpressed in soluble form. Overexpression was achieved without isopropyl beta-d-thiogalactoside induction, and cells grown to stationary phase yielded approximately 30% of target proteins to total soluble cell proteins. Enzyme activity measurements using coupled assays demonstrated that there was a 328-fold increase in specific activity for shikimate kinase and 101-fold increase for EPSP synthase.